Multiplexed Genome Editing

ABSTRACT

A method of modulating some or all copies of a gene in a cell is provided including introducing into a cell one or more ribonucleic acid (RNA) sequences that comprise a portion that is complementary to all or a portion of each of the one or more target nucleic acid sequences, and a nucleic acid sequence that encodes a Cas protein and maintaining the cells under conditions in which the Cas protein is expressed and the Cas protein binds and modulates the one or more target nucleic acid sequences in the cell.

RELATED APPLICATION DATA

This application claims priority to U.S. Provisional Application No.62/239,239 filed on Oct. 8, 2015 which is hereby incorporated herein byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Genome editing via sequence-specific nucleases is known. Anuclease-mediated double-stranded DNA (dsDNA) break in the genome can berepaired by two main mechanisms: Non-Homologous End Joining (NHEJ),which frequently results in the introduction of non-specific insertionsand deletions (indels), or homology directed repair (HDR), whichincorporates a homologous strand as a repair template. See reference 4hereby incorporated by reference in its entirety. When asequence-specific nuclease is delivered along with a homologous donorDNA construct containing the desired mutations, gene targetingefficiencies are increased by 1000-fold compared to just the donorconstruct alone.

Alternative methods have been developed to accelerate the process ofgenome modification by directly injecting DNA or mRNA of site-specificnucleases into the one cell embryo to generate DNA double strand break(DSB) at a specified locus in various species. DSBs induced by thesesite-specific nucleases can then be repaired by either error-pronenon-homologous end joining (NHEJ) resulting in mutant mice and ratscarrying deletions or insertions at the cut site. If a donor plasmidwith homology to the ends flanking the DSB is co-injected, high-fidelityhomologous recombination can produce animals with targeted integrations.Because these methods require the complex designs of zinc fingernucleases (ZNFs) or Transcription activator-like effector nucleases(TALENs) for each target gene and because the efficiency of targetingmay vary substantially, no multiplexed gene targeting has been reportedto date.

Thus, improved methods for producing genetically modified cells togenerate animals, such as pigs, are needed for potential sources oforgans for transplantation.

SUMMARY OF THE INVENTION

Described herein is the use of the Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) and CRISPR associated (Cas) proteins(CRISPR/Cas) system to achieve highly efficient and simultaneoustargeting of multiple nucleic acid sequences in cells.

Aspects of the present disclosure are directed to the modification ofgenomic DNA, such as multiplex modification of DNA, in a cell (e.g.,stem cell, somatic cell, germ line cell, zygote) using one or more guideRNAs (ribonucleic acids) to direct an enzyme having nuclease activityexpressed by the cell, such as a DNA binding protein having nucleaseactivity, to a target location on the DNA (deoxyribonucleic acid)wherein the enzyme cuts the DNA and an exogenous donor nucleic acid isinserted into the DNA, such as by homologous recombination. Aspects ofthe present disclosure include cycling or repeating steps of DNAmodification in a cell to create a cell having multiple modifications ofDNA within the cell. Modifications can include insertion of exogenousdonor nucleic acids. Modifications can include deletion of endogenousnucleic acids.

Multiple nucleic acid sequences can be modulated (e.g., inactivated) bya single step of introducing into a cell, which expresses an enzyme, andnucleic acids encoding a plurality of RNAs, such as byco-transformation, wherein the RNAs are expressed and wherein each RNAin the plurality guides the enzyme to a particular site of the DNA, theenzyme cuts the DNA. According to this aspect, many alterations ormodification of the DNA in the cell are created in a single cycle.

According to one aspect, the cell expressing the enzyme has beengenetically altered to express the enzyme such as by introducing intothe cell a nucleic acid encoding the enzyme and which can be expressedby the cell. In this manner, aspects of the present disclosure includecycling the steps of introducing RNA into a cell which expresses theenzyme, introducing exogenous donor nucleic acid into the cell,expressing the RNA, forming a co-localization complex of the RNA, theenzyme and the DNA, and enzymatic cutting of the DNA by the enzyme.Insertion of a donor nucleic acid into the DNA is also provided herein.Cycling or repeating of the above steps results in multiplexed geneticmodification of a cell at multiple loci, i.e., a cell having multiplegenetic modifications.

According to certain aspects, DNA binding proteins or enzymes within thescope of the present disclosure include a protein that forms a complexwith the guide RNA and with the guide RNA guiding the complex to adouble stranded DNA sequence wherein the complex binds to the DNAsequence. According to one aspect, the enzyme can be an RNA guided DNAbinding protein, such as an RNA guided DNA binding protein of a Type IICRISPR System that binds to the DNA and is guided by RNA. According toone aspect, the RNA guided DNA binding protein is a Cas9 protein.

This aspect of the present disclosure may be referred to asco-localization of the RNA and DNA binding protein to or with the doublestranded DNA. In this manner, a DNA binding protein-guide RNA complexmay be used to cut multiple sites of the double stranded DNA so as tocreate a cell with multiple genetic modifications, such as disruption ofone or more (e.g., all) copies of a gene.

According to certain aspects, a method of making multiple alterations totarget DNA in a cell expressing an enzyme that forms a co-localizationcomplex with RNA complementary to the target DNA and that cleaves thetarget DNA in a site specific manner is provided including (a)introducing into the cell a first foreign nucleic acid encoding one ormore RNAs complementary to the target DNA and which guide the enzyme tothe target DNA, wherein the one or more RNAs and the enzyme are membersof a co-localization complex for the target DNA, wherein the one or moreRNAs and the enzyme co-localize to the target DNA, the enzyme cleavesthe target DNA to produce altered DNA in the cell, and repeating step(a) multiple times to produce multiple alterations to the DNA in thecell.

In some aspects, a method of inactivating expression of one or moretarget nucleic acid sequences in a cell comprises introducing into acell one or more ribonucleic acid (RNA) sequences that comprise aportion that is complementary to all or a portion of each of the one ormore target nucleic acid sequences, and a nucleic acid sequence thatencodes a Cas protein; and maintaining the cells under conditions inwhich the Cas protein is expressed and the Cas protein binds andinactivates the one or more target nucleic acid sequences in the cell.

In other aspects, a method of modulating one or more target nucleic acidsequences in a cell comprises introducing into the cell a nucleic acidsequence encoding an RNA complementary to all or a portion of a targetnucleic acid sequence in the cell; introducing into the cell a nucleicacid sequence encoding an enzyme that interacts with the RNA and cleavesthe target nucleic acid sequence in a site specific manner; andmaintaining the cell under conditions in which the RNA binds tocomplementary target nucleic acid sequence forming a complex, andwherein the enzyme binds to a binding site on the complex and modulatesthe one or more target nucleic acid sequences.

In the methods described herein, the introducing step can comprisetransfecting the cell with the one or more RNA sequences and the nucleicacid sequence that encodes the Cas protein.

In some embodiments, the one or more RNA sequences, the nucleic acidsequence that encodes the Cas protein, or a combination thereof areintroduced into a genome of the cell.

In some embodiments, the expression of the Cas protein is induced.

In the methods described, herein the cell is from an embryo. The cellcan be a stem cell, zygote, or a germ line cell. In embodiments wherethe cell is a stem cell, the stem cell is an embryonic stem cell orpluripotent stem cell. In other embodiments, the cell is a somatic cell.In embodiments, where the cell is a somatic cell, the somatic cell is aeukaryotic cell or prokaryotic cell. The eukaryotic cell can be ananimal cell, such as from a pig, mouse, rat, rabbit, dog, horse, cow,non-human primate, human.

The one or more target nucleic acid sequences can comprise a porcineendogenous retrovirus (PERV) gene. For example, the PERV gene cancomprise a pol gene.

The methods described herein can inactivate, modulate, or effect one ormore copies of the pol gene. In some embodiments, all copies of the polgene in the cell are inactivated.

In some embodiments, the Cas protein is a Cas9.

In some embodiments, the one or more RNA sequences can be about 10 toabout 1000 nucleotides. For example, the one or more RNA sequences canbe about 15 to about 200 nucleotides.

In some aspects an engineered cell comprises one or more endogenousviral genes; and one or more exogenous nucleic acid sequences thatcomprise a portion that is complementary to all or a portion of one ormore target nucleic acid sequences of the one or more endogenous viralgenes; wherein each of the one or more endogenous viral genes of thecell are modulated.

In another aspect, an engineered cell can comprise a plurality ofendogenous retroviral genes; and one or more exogenous nucleic acidsequences that comprise a portion that is complementary to all or aportion of one or more target nucleic acid sequences of the plurality ofendogenous viral genes; wherein each of the plurality of endogenousviral genes of the cell are modulated.

The engineered cells described herein can comprise a porcine endogenousretrovirus (PERV) gene. For example, the PERV gene can comprise a polgene.

In some aspects, modulation of the pol gene inactivates one or morecopies of the pol gene. For example, all or substantially all copies ofthe pol gene in the cell are inactivated.

According to one aspect, the RNA is between about 10 to about 1000nucleotides. According to one aspect, the RNA is between about 20 toabout 100 nucleotides.

According to one aspect, the one or more RNAs is a guide RNA. Accordingto one aspect, the one or more RNAs is a tracrRNA-crRNA fusion.

According to one aspect, the DNA is genomic DNA, mitochondrial DNA,viral DNA, or exogenous DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate CRISPR-Cas9 gRNAs were designed to specificallytarget the pol gene in 62 copies of PERVs in PK15 cells. (A)Phylogenetic tree representing endogenous retroviruses present in thepig genome. PERVs are highlighted in blue. (B) Copy number determinationof PERVs in PK15 cells via digital droplet PCR. The copy number of polelements was estimated to be 62 using three independent reference genes:ACTB, GAPDH, and EB2. N=3, mean+/−SEM. (C) Two CRISPR-Cas9 gRNAs weredesigned to target the catalytic region of the PERV pol gene. The twogRNA targeting sequences are shown below a schematic of PERV genestructure. Their PAM sequences are highlighted in red. (SEQ ID NO:27-28)

FIGS. 2A-2B illustrate clonal PK15 cells with inactivation of all copiesof PREV pol genes after Cas9 treatment. (A) A bimodal distribution ofpol targeting efficiencies was observed among the single-cell-derivedPK15 clones after 17 days of Cas9 induction. 45/50 exhibited <16%targeting efficiency; 5/50 clones exhibited >93% targeting efficiency.(B) PK15 haplotypes at PERV pol loci after CRISPR-Cas9 treatment. Inred, indel events in the PERV pol sequence are represented. Shades ofpurple indicate endogenous PERVs.

FIGS. 3A-3D illustrate: (A) Detection of PERV pol, gag, and env DNA inthe genomes of HEK-293-GFP cells after co-culturing with PK15 cells for5 days and 7 days (293G5D and 393G7D, respectively). A pig GGTA1 primerset was used to detect pig cell contamination of the purified humancells. (B) qPCR quantification of the number of PERV elements in 1000293G cells derived from a population co-cultured with wild type PK15cells using specific primer sets. (N=3, mean+/−SEM) (C) qPCRquantification of the number of PERV elements in PK15 Clones 15, 20, 29,and 38, with high levels of PERV pol modification, and minimallymodified Clones 40 and 41. (N=3, mean+/−SEM) (D) Results of PCR on PERVpol on genomic DNA from various numbers of HEK 293-GFP cells (0.1, 1,10,and 100) isolated from populations previously cultured with highlymodified PK15 Clone 20 and minimally modified Clone 40. See FigureS18-21 for a full panel of PCR reactions.

FIG. 4 (S1) illustrates PERV pol consensus sequence and gRNA design.

FIG. 5 (S2) is a schematic of CRISPR/Cas9 construct targeting PERVs.

FIG. 6 (S3) illustrates measurement of Cas9-gRNAs activity.

FIG. 7 (S4) illustrates optimization of DOX concentration to induce Cas9expression for PERV targeting.

FIG. 8 (S5) illustrates time series measurement of Piggybac-Cas9/gRNAsPERV targeting efficiencies.

FIG. 9 (S6) illustrates time series measurement of Lenti-Cas9/2gRNAsPERV targeting efficiency.

FIG. 10 (S7) illustrates Sanger sequencing validation of PERV targetingefficiency and indel patterning. (SEQ ID NO:29)

FIG. 11 (S8) illustrates repeated the gene editing experiment.

FIGS. 12A-B (S9) illustrates PERV pol targeting efficiency of singlecells.

FIG. 13 (S10) illustrates phylogeny of PERV haplotypes.

FIG. 14 (S11) illustrates distribution of pol gene disruption.

FIGS. 15A-15B (S12) illustrate karyotype analysis of highly and lowlymodified PK15 clones.

FIG. 16 (S13) illustrates Summary of karyotype analysis of PK15 clones.

FIG. 17 (S14) illustrates Karyotype nomenclature.

FIG. 18 (S15) illustrates Detection of PERV reverse transcriptaseactivity.

FIG. 19 (S16) illustrates an experimental design to detect thetransmission of PERVs to human cells.

FIGS. 20A-20C (S17) illustrate quality control of the purifiedHEK293-GFP cells by FACS.

FIGS. 21A-21D (S18) illustrates detection of pig cell contamination inHEK293 cells using pig GGTA1 primers.

FIGS. 22A-22D (S19) illustrates detection of PERV DNA elements in HEK293cells using PERV pol primers.

FIGS. 23A-23D (S20) illustrates detection of PERV DNA elements in HEK293cells using PERV env primers.

FIGS. 24A-24D (S21) illustrates detection of PERV DNA elements in HEK293cells using PERV gag primers.

FIGS. 25A-25B (S22) illustrates Cas9/2gRNAs expression levels in highlyand lowly modified clones.

FIG. 26 (S23) illustrates principle component analysis of highly andlowly modified PK15 clones.

FIGS. 27A-27B (S24) illustrate gene set enrichment analysis.

FIG. 28 (S25) illustrates indel composition analysis and comparisonamong highly modified clones.

FIGS. 29A-29D (S26) illustrates a Markov model analysis of DNA repairprocesses leading to Cas9 elimination of active PERV elements.

FIG. 30 (S27) illustrates off-target analysis using Whole GenomeSequencing (WGS).

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention are directed to the use of CRISPR/Cas9,for nucleic acid engineering. Described herein is the development of anefficient technology for the generation of animals (e.g., pigs) carryingmultiple mutated genes. Specifically, the clustered regularlyinterspaced short palindromic repeats (CRISPR) and CRISPR associatedgenes (Cas genes), referred to herein as the CRISPR/Cas system, has beenadapted as an efficient gene targeting technology e.g., for multiplexedgenome editing. Demonstrated herein is that CRISPR/Cas mediated geneediting allows the simultaneous inactivation of 62 copies of the porcineendogenous retrovirus (PERV) pol gene in a porcine kidney epithelialcell line (e.g., PK15) with high efficiency. Co-injection ortransfection of Cas9 mRNA and guide RNA (gRNA) targeting PERVs intocells generated a greater than 1000 fold reduction in PERV transmissionto human cells with biallelic mutations in both genes with an efficiencyof up to 100%. Shown herein is that the CRISPR/Cas system allows the onestep generation of cells carrying inactivation of all copies of PERV. Incertain embodiments a method described herein generates cell andanimals, e.g., pigs, with inactivation of 1, 2, 3, 4, 5, or more geneswith an efficiency of between 20% and 100%, e.g., at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more, e.g., up to 96%, 97%,98%, 99%, or more.

EXEMPLIFICATION Example 1 Genome-Wide Inactivation of Porcine EndogenousRetroviruses (PERVs)

The shortage of organs for transplantation is a major barrier to thetreatment of organ failure. While porcine organs are consideredpromising, their use has been checked by concerns about transmission ofporcine endogenous retroviruses (PERVs) to humans. Here, the eradicationof all PERVs in a porcine kidney epithelial cell line (PK15) wasperformed. It was first determined the PK15 PERV copy number to be 62.Using CRISPR-Cas9, all 62 copies of the PERV pol gene were disrupted anddemonstrated a >1000-fold reduction in PERV transmission to human cellsusing our engineered cells. This study showed that CRISPR-Cas9multiplexability can be as high as 62 and demonstrates the possibilitythat PERVs can be inactivated for clinical application toporcine-to-human xenotransplantation.

Pig genomes contain from a few to several dozen copies of PERV elements.Unlike other zoonotic pathogens, PERVs cannot be eliminated by biosecurebreeding. Prior strategies for reducing the risk of PERV transmission tohumans have included small interfering RNAs (RNAi), vaccines, and PERVelimination using zinc finger nucleases and TAL effector nucleases, butthese have had limited success. Here, the successful use of theCRISPR-Cas9 RNA-guided nuclease system can be used to inactivate allcopies of the PERV pol gene and effect a 1000-fold reduction of PERVinfectivity of human cells.

To design Cas9 guide RNAs (gRNAs) that specifically target PERVs, thesequences of publically available PERVs and other endogenousretroviruses in pigs (Methods) were analyzed. A distinct clade of PERVelements (FIG. 1A) were identified and determined there to be 62 copiesof PERVs in PK15 cells using droplet digital PCR (FIG. 1B). Two Cas9guide RNAs (gRNAs) were designed that targeted the highly conservedcatalytic center of the pol gene on PERVs (FIG. 1C, Fig. S1). The polgene product functions as a reverse transcriptase (RT) and is thusessential for viral replication and infection. It was determined thatthese gRNAs targeted all PERVs but no other endogenous retrovirus orother sequences in the pig genome (Methods).

Initial experiments showed inefficient PERV editing when Cas9 and thegRNAs were transiently transfected (Fig. S2). Thus a PiggyBac transposonsystem was used to deliver a doxycycline-inducible Cas9 and the twogRNAs into the genome of PK15 cells (Fig. S2-3). Continuous induction ofCas9 led to increased targeting frequency of the PERVs (Fig. S5), with amaximum targeting frequency of 37% (˜23 PERV copies per genome) observedon day 17 (Fig. S5). Neither higher concentrations of doxycycline orprolonged incubation increased targeting efficiency (Fig. S4,5),possibly due to the toxicity of non-specific DNA damage by CRISPR-Cas9.Similar trends were observed when Cas9 was delivered using lentiviralconstructs (Fig. S6). The cell lines that exhibited maximal PERVtargeting efficiencies were genotyped. 455 different insertion anddeletion (indel) events centered at the two gRNA target sites (FIG. 2B)was observed. Indel sizes ranged from 1 to 148 bp; 80% of indels weresmall deletions (<9 bp). The initial deep sequencing results wasvalidated with Sanger Sequencing (Fig. S7).

Single cells from PK15 cells with high PERV targeting efficiency weresorted using flow cytometry and genotyped the pol locus of the resultingclones via deep sequencing. A repeatable bimodal (FIG. 2A, S8-9)distribution was observed with ˜10% of the clones exhibiting high levelsof PERV disruption (97%-100%), and the remaining clones exhibiting lowlevels of editing (<10%). Individual indel events were examined in thegenomes of these clones (FIG. 2B, Fig. S10-11). For the highly editedclones (Clone 20, 100%; Clone 15, 100%; Clone 29, 100%; Clone 38,97.37%), only 16-20 unique indel patterns in each clone (FIG. 2B, S11)were observed. In addition, there was a much higher degree of repetitionof indels within each clone than across the clones (Fig. S25),suggesting a mechanism of gene conversion in which previously mutatedPERV copies were used as templates to repair wild-type PERVs cleaved byCas9 (FIG. 2B, Fig. S25). Mathematical modeling of DNA repair duringPERV elimination (Fig. S26) and analysis of expression data (Fig.S22-24) supported this hypothesis and suggested that highly editedclones were derived from cells in which Cas9 and the gRNAs were highlyexpressed.

Next, unexpected genomic rearrangements had occurred as a result of themultiplexed genome editing was examined. Karyotyping of individualmodified clones (Fig. S12-S14) indicated that there were no observablegenomic rearrangements. 11 independent genomic loci with at most 2 bpmismatches to each of the intended gRNA targets were examined andobserved no non-specific mutations (Fig. S27). This suggests that ourmultiplexed Cas9-based genome engineering strategy did not causecatastrophic genomic instability.

Last, disruption of all copies of PERV pol in the pig genome couldeliminate in vitro transmission of PERVs from pig to human cells wasexamined. No detection of RT activity in the cell culture supernatant ofthe highly modified PK15 clones (Fig. S15) was observed, suggesting thatmodified cells only produced minimal amounts of PERV particles.Co-culture of WT and highly modified PK15 cells with HEK 293 cells weretested directly for transmission of PERV DNA to human cells. Afterco-culturing PK15 WT and HEK 293 cells for 5 days and 7 days (Fig.S16-17), PERV pol, gag, and env sequences in the HEK 293 cells weredetected (FIG. 3A). The estimated frequency of PERV infection wasapproximately 1000 PERVs/1000 human cells (FIG. 3B). However, PK15clones with >97% PERV pol targeting exhibited up to 1000-fold reductionof PERV infection, similar to background levels (FIG. 3C). These resultswere validated with PCR amplification of serial dilutions of HEK293cells that had a history of contact with PK15 clones (FIG. 3D, S18-21).PERVs in single HEK293 cells isolated from the population co-culturedwith minimally modified Clone 40 was consistently detected, but couldnot distinctly detect PERVs in 100 human cells from the populationco-cultured with highly modified Clone 20. Thus, PERV infectivity of theengineered PK15 cells had been reduced by up to 1000 fold.

In summary, it was successfully targeted the 62 copies of PERV pol inPK15 cells and demonstrated greatly reduced in vitro transmission ofPERVs to human cells. While in vivo PERV transmission to humans has notbeen demonstrated, PERVs are still considered risky and our strategycould completely eliminate this. As no porcine embryonic stem cellsexist, this system will need to be recapitulated in primary porcinecells and cloned into animals using somatic cell nuclear transfer.Moreover, simultaneous Cas9 targeting of 62 loci in single pig cellswithout salient genomic rearrangement was achieved. To our knowledge,the maximum number of genomic sites previously reported to besimultaneously edited has been six. Our methods thus open thepossibility of editing other repetitive regions of biologicalsignificance.

Example 2 Methods

PERV copy number quantification: Droplet Digital PCR™ PCR (ddPCR™) wasusd to quantify the copy number of PERVs according to the manufacturer'sinstructions (Bio-Rad). Briefly, genomic DNA (DNeasy Blood & Tissue Kit,Qiagen) from cultured cells was purified, digested 50 ng genomic DNAwith MseI (10U) at 37° C. for 1 hour, and prepared the ddPCR reactionwith 10 μl 2X ddPCR Master mix, 1 μl of 18 μM target primers & 5 μMtarget probe (VIC), 1 μl of 18 μM reference primers & 5 μM referenceprobe (FAM), 5 ng digested DNA, and water to total volume of 20 μl. Thesequence of the primers and the probe information can be found inExtended Data Table 1.

Methods

TABLE 1 Primers used in ddPCR assay Name Sequence PrimerPol1-FWCGACTGCCCCAAGGGTTCAA (SEQ ID NO: 1) PrimerPol2-FW CCGACTGCCCCAAGAGTTCAA(SEQ ID NO: 2) PrimerPol-RV TCTCTCCTGCAAATCTGGGCC (SEQ ID NO: 3)ProbePol /56FAM/CACGTACTGGAGGAGGGTCACCTG (SEQ ID NO: 4)Primerpig_actin_F Taaccgatcctttcaagcattt (SEQ ID NO: 5)Primerpig_actin_R Tggtttcaaagcttgcatcata (SEQ ID NO: 6) Probepig_actin/5Hex/cgtggggatgcttcctgagaaag (SEQ ID NO: 7) Primerpig_GAPDH_FCcgcgatctaatgttctctttc (SEQ ID NO: 8) Primerpig_GAPDH_RTtcactccgaccttcaccat (SEQ ID NO: 9) Probepig_GAPDH/5Hex/cagccgcgtccctgagacac (SEQ ID NO: 10)

CRISPR-Cas9 gRNAs design: MUSCLE was used to carry out a multiplesequence alignment of 245 endogenous retrovirus found in the porcinegenome. A phylogenetic tree of the sequences was built and identified aclade that included the PERVs (see FIG. 1a ). The R library DECIPHER wasused to design specific gRNAs that target all PERVs but no otherendogenous retroviral sequences.

Cell culture: PK15 were maintained in Dulbecco's modified Eagle's medium(DMEM, Invitrogen) high glucose supplemented with 10% fetal bovine serum(Invitrogen), and 1% penicillin/streptomycin (Pen/Strep, Invitrogen).All cells were maintained in a humidified incubator at 37° C. and 5%CO₂.

PiggyBac-Cas9/2gRNAs construction and cell line establishment:PiggyBac-Cas9/2gRNAs construct is derived from a plasmid previouslyreported in Wang et al (2). Briefly, a DNA fragment encodingU6-gRNA1-U6-gRNA2 was synthesized (Genewiz) and incorporated it into aPiggBac-Cas9 construct. To establish PK15 cell lines withPiggyBac-Cas9/2gRNAs integration, 5·10⁵ PK15 cells was transfected with4 μg PiggyBac-Cas9/2gRNAs plasmid and 1 μg Super PiggyBac Transposaseplasmid (System Biosciences) using Lipofectamine 2000 (Invitrogen). Toenrich for the cells carrying the integrated construct, 2 μg/mLpuromycin was added to the transfected cells. Based on the negativecontrol, puromycin was applied to wild type PK15 cells, it wasdetermined that the selection completed in 3 days. The PK15-PiggyBaccell lines were maintained with 2 μg/mL puromycin hereafter. 2 μg/mldoxycycline was applied to induce Cas9 expression.

Lentivirus-Cas9/2gRNAs construction and cell line establishment:Lenti-Cas9/2gRNAs constructs were derived from a plasmid previouslyreported (3). A DNA fragment encoding U6-gRNA1-U6-gRNA2 was synthesized(Genewiz) and incorporated it into a Lenti-Cas9-V2. To generatelentivirus carrying Lenti-Cas9/2gRNAs, ˜5·10⁶ 293FT HEK cells wastransfected with 3 μg Lenti-Cas9-gRNAs and 12 μg ViraPower LentiviralPackaging Mix (Invitrogen) using Lipofectamine 2000. The lentiviralparticles were collected 72 hours after transfection, and the viraltiter was measured using Lenti-X GoStix (Takara Clonetech). ˜10⁵lentiviral particles to ˜1·10⁶ PK15 cells were transduced and conductedselection by puromycin to enrich transduced cells 5 days aftertransduction. The PK15-Lenti cell lines were maintained with 2 μg/mLpuromycin thereafter.

Genotyping of colonized and single PK15 cells: PK15 cultures weredissociated using TrypLE (Invitrogen) and resuspended in PK15 mediumwith the viability dye ToPro-3 (Invitrogen) at a concentration of1−2·10⁵ cells/ml. Live PK15 cells were single-cell sorted using a BDFACSAria II SORP UV (BD Biosciences) with 100 mm nozzle under sterileconditions. SSC-H versus SSC-W and FSC-H versus FSC-W doubletdiscrimination gates and a stringent ‘0/32/16 single-cell’ sorting maskwere used to ensure that one and only one cell was sorted per well.Cells were sorted in 96-well plates with each well containing 100μl PK15medium. After sorting, plates were centrifuged at 70 g for 3 min. Colonyformation was seen 7 days after sorting and genotyping experiment wasperformed 2 weeks after FACS.

To genotype single PK15 cells without clonal expansion, the PERV locuswas directly amplified from sorted single cells according to apreviously reported single cell genotyping protocol (4). Briefly, priorto sorting, all plastics and non-biologic buffers were treated with UVradiation for 30 min. Single cells were sorted into 96-well PCR plateswith each well carrying 0.5 μl 10X KAPA express extract buffer (KAPABiosystems), 0.1 μl of 1 U/μl KAPA Express Extract Enzyme and 4.6 μlwater. The lysis reaction was incubated at 75° C. for 15 min andinactivated the reaction at 95° C. for 5 min. All reactions were thenadded to 25 μl PCR reactions containing 12.5 μl 2X KAPA 2G fast (KAPABiosystems), 100 nM PERV illumina primers (Methods Table2), and 7.5 μlwater. Reactions were incubated at 95° C. for 3 min followed by 25cycles of 95° C., 10 s; 65° C., 20 s and 72° C., 20 s. To add theIllumina sequence adaptors, 5 μl of reaction products were then added to20 μl of PCR mix containing 12.5 ml of 2 KAPA HIFI Hotstart Readymix(KAPA Biosystems), 100 nM primers carrying Illumina sequence adaptorsand 7μl water. Reactions were incubated at 95° C. for 5 min followed by15-25 cycles of 98° C., 20 s; 65° C., 20 s and 72° C., 20 s. PCRproducts were checked on EX 2% gels (Invitrogen), followed by therecovery of 300-400 bp products from the gel. These products were thenmixed at roughly the same amount, purified (QIAquick Gel ExtractionKit), and sequenced with MiSeq Personal Sequencer (Illumina). Deepsequencing data was analyzed and determined the PERV editing efficiencyusing CRISPR-GA (5).

TABLE 2 Primers used in the PERV pol genotyping Name Sequence illumina_ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGACT primerPol1GCCCCAAGGGTTCAA (SEQ ID NO: 11) illumina_ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGAC primerPol2TGCCCCAAGAGTTCAA (SEQ ID NO: 12) illumina_GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTC primerPo3TCCTGCAAATCTGGGCC (SEQ ID NO: 13)

Targeting efficiency estimation: a custom pipeline was built to estimatethe efficiency of PERV inactivation. Briefly, the pol gene was amplifiedand sequenced via Illumina Next Generation Sequencing using PE250 orPE300. First, the two overlapping reads were combined using PEAR (6) andmapped to the reference region using BLAT. After mapping, the reads weregrouped into sets containing specific combinations of haplotypes (seeExtended Data FIG. 7), and indel types. Read sets with representationlower than 0.5% of the total number of mapped reads were discarded.Finally, the mapping output was parsed to call the different insertionsand deletions as described in Güell et al (5).

RNA-seq analysis: The susScr3 pig genome and Ensembl transcripts wereobtained from the UCSC Genome Brower Database. RNA-Seq reads were mappedto the reference genome using the STAR software (7) and the RPKM of thetranscripts were quantified using BEDTools (8). Differential expressionanalysis was performed in R using the DESeq2 package (9), and gene setenrichment analysis was carried out by the GSEA software (10), with geneset definitions obtained from the software's website.

Reverse transcriptase (RT) assay: To test the RT activity of the PK15cells and modified PK15 clones (4 highly and 1 lowly modified clones),5·10⁵ cells were plated in T75 cm² flasks, and collected the supernatant4 days after seeding. The media was filtered using a 0.45 μM Millex-HVSyringe Filter (EMD Millipore Corporation), and the filtered supernatantwas concentrated at 4000 g for 30 min using Amicon Ultra-15 CentrifugalFilter Unit (EMD Millipore Corporation). The concentrated supernatantwas ultra-centrifuged at 50,000 rpm for 60 min. The supernatant wascarefully removed, and the virus pellet was collected and lysed with 20μl of 10% NP40 at 37° C. for 60 min.

The RT reaction was conducted using the Omniscript RT Kit (Qiagen). Thetotal volume of the reaction was 20 μl, which contained 1□ RT buffer,0.5 mM dNTPs, 0.5 μM Influenza reverse primer (5′ CTGCATGACCAGGGTTTATG3′) (SEQ ID NO:14), 100 units of RnaseOUT (Life Technology, Invitrogen),100 units of SuperRnase Inhibitor (Life Technologies), 5 μl of samplelysis and 40 ng of IDT-synthesized Influenza RNA template which wasrnase resistant in both 5′ and 3′ end. The RNA template sequence was 5′rA*rA*rC*rA*rU*rGrGrArArCrCrUrUrUrGrGrCrCrCrUrGrUrUrCrArUrUrUrUrArGrArArArUrCrArArGrUrCrArArGrArUrArCrGrCrArGrArArGrArGrUrArGrArCrArUrArArArCrCrCrUrGrGrUrCrArUrGrCrArGrArCrCrU*rC*rA*rG*rU*rG 3′ (* phosphodiester bond)(SEQ ID NO:15). After the RT reaction was completed, the RT product wasexamined by PCR using Influenza forward (5′ ACCTTTGGCCCTGTTCATTT 3′)(SEQ ID NO:16) and Influenza reverse primers (sequence shown as above).The expected size of the amplicon was 72 bp.

Infectivity Assay

HEK293-GFP cell line establishment: The Lenti-GFP construct was derivedfrom the plasmid pLVX-IRES-ZsGreenl (Clontech. Catalog No. 632187;PT4064-5). To generate the lentivirus carrying Lenti-GFP, ˜5·10⁶ 293FTHEK cells were transfected with 3 μg of pVX-ZsGreen plasmid and 12 μg ofViraPower Lentiviral Packaging Mix (Invitrogen) using Lipofectamine 2000(Invitrogen). Lentiviral particles were collected 72 hours aftertransfection, and the viral titer was measured using Lenti-X GoStix(Takara Clonetech). ˜10⁵ lentivirus particles to ˜1·10⁶ HEK293 cellswere transfected and conducted selection by puromycin to enrich thetransduced cells 5 days after transduction. The 293-GFP-Lenti cell lineswere maintained with 0.5 μg/mL puromycin thereafter.

Infectivity test of PK15 WT to HEK293-GFP: 1·10⁵ cells ofLenti-GFP-293FT HEK cells and 1·10⁵ PK15 WT cells were cultured togetherin a 6-well plate. In parallel, 2·10⁵ PK15 WT cells were cultured alonein another well as a control. The puromycin selection experiment wasdone by adding 5 μg/ml of the antibiotic for 7 days. The time point wasdetermined when no viable cells in the control well and approximately100% GFP positive cells in the experimental well as the time point whenthe puromycin selection was completed to purify lenti-GFP-293FT humancells. Cells from the 293FT HEK/PK15 WT co-culture were collected atdifferent time periods. The genomic DNA was extracted using (DNeasyBlood & Tissue Kit, Qiagen) from cultured cells of the 293-GFP WT, PK15WT and the co-cultured cells. The genomic DNA concentration was measuredusing a Qubit 2.0 Fluorometer (Invitrogen), and 3 ng from each samplewas used as DNA template for PCR. In all, 1 μL of the genomic DNA wereadded to 25 μL of a PCR mix containing 12.5 μL 2X KAPA Hifi HotstartReadymix (KAPA Biosystems) and 100 μM of primers as listed in MethodsTable 3. Reactions were incubated at 95° C. for 5 min followed by 35cycles of 98° C., 20 s; 65° C., 20 s and 72° C., 20 s. PCR products werevisualized on EX 2% gels (Invitrogen) and observed for bands of 300-400base pairs.

TABLE 3 A table exhibiting the primers used in the infectivity assayName Sequence PERV pol-Forward GGG AGT GGG ACG GGT AAC CCA(SEQ ID NO: 17) PERV pol-Reverse GCC CAG GCT TGG GGA AAC TG(SEQ ID NO: 18) PERV env-Forward ACC TCT TCT TGT TGG CTT TG(SEQ ID NO: 19) PERV env-Reverse CAA AGG TGT TGG TGG GAT GG(SEQ ID NO: 20) PERV gag-Forward CGC ACA CTG GTC CTT GTC GAG(SEQ ID NO: 21) PERV gag-Reverse TGA TCT AGT GAG AGA GGC AGA G(SEQ ID NO: 22) Pig GGTA1-Forward GGA GCC CTT AGG GAC CAT TA(SEQ ID NO: 23) Pig GGTA1-Reverse GCG CTA AGG AGT GCG TTC TA(SEQ ID NO: 24) Human ACTB-Forward GCC TTC CTT CCT GGG CAT GG(SEQ ID NO: 25) Human ACTB-Reverse GAG TAC TTG CGC TCA GGA GG(SEQ ID NO: 26)

Quantification of PERV copy numbers infected in HEK293-GFP cells: qPCRwas performed to quantify the PERV copy number in HEK293-GFP cells.Genomic DNA of PK15 WT cells of different amounts was used as thetemplate for the qPCR reactions. Reactions were conducted in triplicateusing KAPA SYBR FAST qPCR Master Mix Universal (KAPA Biosystems). PERVpol, env, gag primers, human ACTB and pig GGTA1 primers (Methods Table3) were added to a final concentration of 1 μM. Reactions were incubatedat 95° C. for 3 min (enzyme activation) followed by 50 cycles of 95° C.,5 s (denaturation); 60° C., 60 s (annealing/extension). The logarithm ofthe genomic DNA amount linearizes with the quantification cycle (Cq).pol, gag, env primers were used to examine for presence of PERVs. PigGGTA1 primers served to control for potential porcine genomecontaminants in human cells after infection. All experiments wereconducted in triplicate.

Infectivity Assay of the Modified PK15 clones to HEK293-GFP: 1·10⁵ cellsof HEK293-GFP cells and 1·10⁵ cells of the high modified (15, 20, 29,38) clones and low modified clones (40, 41) were co-cultured in a 6-wellplate for 7 days. To isolate the HEK293-GFP cells in order to examinefor PERV elements, the GFP positive cells were double sorted to purifythe human cell populations.

To quantify the PERV infectivity of different clones to HEK293-GFPcells, both qPCR assays and PCR assays were conducted on series dilutedHEK293-GFP cells after sorting. For the qPCR assays, the genomic DNA(DNeasy Blood & Tissue Kit, Qiagen) was extracted from double sortedHEK293-GFP cells. The genomic DNA concentration was measured using Qubit2.0 fluorometer (Invitrogen). In all, 3 ng of the genomic DNA was addedto 20 μL of KAPA SYBR FAST qPCR reaction (KAPA Biosystems) using PERVpol, env, gag and pig GGTA primers respectively (Extended Data Table 2).The qPCR procedure was performed as described above. For the seriesdilution assay, purified HEK293-GFP cells were sorted (1 cell/well, 10cells/well, 100 cells/well, 1000 cells/well) into 96-well PCR plates fordirect genomic DNA extraction and PCR reactions. Briefly, cells weresorted into 20 μL lysis reaction including 2 μL of 10X KAPA ExpressExtract Buffer, 0.4 μL of 1 U/μl KAPA Express Extract Enzyme and 17.6 μLof PCR-grade water (KAPA Biosystems). The reactions were then incubatedat 55° C. for 10 min (lysis), then at 95° C. for 5 min (enzymeinactivation). Subsequently, the PCR master mix was prepared. In all, 2μL of the genomic DNA lysis was added to 4 different 25 μL of KAPA HifiHotstart PCR reactions (KAPA Biosystems) using 1 μM PERV pol, env, gagprimers, and pig GGTA primers, respectively (Extended Data Table 2). Thereactions were incubated at 95° C. for 3 min (initial denaturation)followed by 35 cycles of 95° C., 15 s (denaturation); 60° C., 15 s(annealing), 72° C., 15 sec/kb, then 75° C., 1 min/kb (final extension).(KAPA Biosystems). The PCR products were visualized on 96 well E-Gel®Agarose Gels, SYBR® Safe DNA Gel (Invitrogen).

CRISPR-Cas9 off-target analysis: whole genome sequencing (WGS) data wasobtained for PK15 (untreated cell line) and clone 20 (highly editedclone). To investigate potential off-target effects of the Cas9/2gRNAs,the reference sequence (Sus Scrofa 10.2) was searched for sites thatdiffered from the 20 bp sequences targeted by the two gRNAs by only 1 or2 bp. 11 such sites were identified and extracted them, together with200 bp of their neighboring regions (Fig. S1). BLAT was used to map theWGS reads to the extracted reference sequences and searched forpotential indel patterns that had emerged in Clone 20 as a result ofoff-target effects. An average coverage of 7-8 X per loci was obtained.Reads with <50 bp matches with the reference sequence were excluded. Incase of reads that mapped to the reference sequence with multiplealignment blocks, which could indicate the presence of indels, readswhose alignment blocks contained <20 bp matches were excluded with thereference sequence. After inspecting the remaining mapped reads, therewas no detection any off-target indel patterns present in clone 20.Another challenge was to comprehensive searches for off-targets here isthat the Sus Scrofa genome is still neither complete nor completelyassembled, limiting the ability to do whole-genome analysis.

Mathematical model of DNA repair process interaction during cumulativePERV inactivation: In this study PERV elements were inactivated bymutations generated by DNA repair processes in response to dsDNA cutscreated by Cas9. It is generally understood that dsDNA cuts may berepaired either by non-homologous end joining (NHEJ) or HomologousRepair (HR), and that while HR can create precise copies of a DNAtemplate sequence at the cut site given the presence of a template withsuitable homology arms, NHEJ can generate mutations (especially indels)and is often considered “error prone.” However, there is also evidencethat NHEJ can also repair dsDNA cuts highly accurately (11, 12), and therelative rates of mutated vs. perfect repair by NHEJ have never beenprecisely measured. Especially when efficient targeted nucleases such asCas9 are expressed for protracted time periods, perfect repair of a cutsite by either NHEJ or HR would regenerate a target site that could becut again. A plausible hypothesis is that the process of perfect repairand re-cutting would occur repeatedly until a mutation arose thatdestroyed the nuclease's ability to recognize the target site. Toexplore the way these repair modalities might work together during thecourse of PERV elimination, their interactions as a Markov process wasmodeled. Specifically, it was assumed:

-   -   There are N identical copies of the nuclease target in a cell.    -   Only wild-type targets are recognized and cut, and only one        target is cut and repaired at a time.    -   DNA repair is either        -   perfect restoration of the target site by NHEJ (with            probability n)        -   NHEJ that results in generation of a mutation that ablates            target recognition (with probability m)        -   repair by HR using any one of the other N−1 target sequences            in the cell (with probability h)        -   Thus, n+m+h=1.

The Markov model computes the probability distribution P^((c))=(p₀^((c)),p₁ ^((c)), . . . , p_(N) ^((c))), where p_(i) ^((c)) is theprobability that there are i target-ablating mutations at cut c, wherec=0, 1, 2 . . . It is assumed that the initial condition p⁽⁰⁾=(1,0, . .. ,0), i.e., that all targets begin as wild-type. The N+1-by-N+1transition matrix M is given as

${ \begin{matrix}{{M( {i,i} )} = {n + {h \cdot \frac{N - i - 1}{N - 1}}}} \\{{M( {i,{i + 1}} )} = {m + {h \cdot \frac{i}{N - 1}}}}\end{matrix} \} \mspace{14mu} {for}\mspace{14mu} 0} \leq i < N$M(N, N) = 1  for  i = NM(i, j) = 0  for  all  other  0 ≤ i, j ≤ N

Finally, P^((c+1))=p^((c))M for c=0,1,2, . . .

The formulas for M assume proposition ii above and state in mathematicalterms that the number of mutated sites in a cell remains unchangedwhenever a cut at a wild-type site is repaired perfectly by NHEJ or byHR using another copy of the wild-type template (formula for M(i, i)),but increases by one if the cut is repaired by mutagenic NHEJ or by HRusing a previously mutated site (formula for M(i, i +1)).

The model incorporates two notable simplifications to actual biology:(i) Target recognition is assumed to be binary—either the nucleaserecognizes a target or it does not. This is tantamount to assuming thatsmall mutations that still support target recognition do notsubstantially alter wild-type cutting rates and therefore can beeffectively lumped together with wild-type sites. (ii) HR repairs usingmutated vs. wild-type templates are assumed to be equally efficient.Modifications could be made to the model to address thesesimplifications, but this is not considered here. It is also worthnoting that, formally, given assumption ii above, the Markov processshould actually stop should the condition p_(N) ^((c))=1 be reached forsome value of c, since at this point no wild-type sites remain to becut, whereas what happens instead mathematically is that cuts continuebut the model remains in a fixed state. Finally, the model effectivelyrepresents the mutation count distribution as a function of independentvariable c (number of cuts) and not as a function of time. No predictionis made regarding the time rates of DNA repair or PERV site elimination,although time can be assumed to increase monotonically with c.

To analyze PERV elimination through the Markov model, N was always setto 62. However, since the relative efficiencies of perfect vs. mutagenicNHEJ repair are unknown (as noted above), and because relative rates ofmutagenic NHEJ vs. HR repair can vary widely depending on cell state andtype, the mutation count distributions for a discrete grid covering thecomplete two-dimensional space of all possible parameter values for n,m, and h, (2500 parameter combinations in all) was computed. The modelwas implemented both as a MatLab (Mathworks, Waltham) script and as an Rscript using the library markovchain (available as Supplemental FilesmodelMarkov.m, modelMarkov.R, respectively).

In addition to computing the mutation count distribution via the Markovmodel for particular parameter values, the MatLab script performedrandom simulations of the NHEJ and HR repair processes throughout aseries of K cuts, allowing bivariate distributions of the numbers oftotal mutations vs. distinct NHEJ events to be estimated, illustrated inFIG. 27 B-C. The R script was used to estimate the most likely state ofthe system over the grid of n, m, and h combinations described above. Kwas varied depending on the computation. As illustrated in Fig. S27, theinvariable result of the model was a unimodal distribution of mutationcounts whose mean advanced towards fixation at N mutations with c, andin Figures S27 B.C, K was set to a value high enough to demonstratefixation. For the calculation of the most likely state of the systemover the n, m, and h grid, K was set to 50, 100, 200, or 500, and 100simulations were conducted for each parameter combination.

Data Deposition

Illumina Miseq data with PERVs elements genotyping data has beenuploaded to the European Nucleotide Archive (ENA) hosted by the EuropeanBioinformatics Institute (EBI) with the submission reference PRJEB11222.

Appendix A provide further information regarding various aspects of thepresent teachings, which is herein incorporated by reference in itsentirety.

The DNA sequence listing further includes genome sequences of multipleendogenous retroviral elements extracted from pig genome sequence andfrom public sequence databases. (SEQ ID NO:30-280)

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1.-43. (canceled)
 44. A genetically engineered porcine cell in which atleast 97% of the PERV pol genes comprise a genetically engineeredgenomic indel.
 45. The genetically engineered porcine cell of claim 44,wherein 100% of the PERV pol genes comprise a genetically engineeredgenomic indel.
 46. The genetically engineered porcine cell of claim 44,wherein the genetically engineered porcine cell exhibits at least a1000-fold reduction in PERV transmission as evidenced by co-culturing ahuman cell with the genetically engineered porcine cell as compared toco-culturing a human cell with a wild-type porcine cell.
 47. Thegenetically engineered porcine cell of claim 46, wherein the human cellis HEK293.
 48. The genetically engineered porcine cell of claim 44,wherein the genetically engineered genomic indel is observed bysequencing.
 49. The genetically engineered porcine cell of claim 44,wherein the genetically engineered porcine cell contains intact PERV gagand/or env genes.
 50. The genetically engineered porcine cell of claim44, wherein the genetically engineered porcine cell is from an embryo.51. The genetically engineered porcine cell of claim 44, wherein thegenetically engineered porcine cell is a stem cell, zygote, or germ linecell.
 52. The genetically engineered porcine cell of claim 44, whereinthe genetically engineered porcine cell is an embryonic stem cell orpluripotent stem cell.
 53. The genetically engineered porcine cell ofclaim 44, wherein the genetically engineered porcine cell is a somaticcell.
 54. A progeny cell derived from the genetically engineered porcinecell of claim
 44. 55. A composition comprising a plurality of theprogeny cell of claim
 54. 56. An isolated porcine organ comprising aplurality of genetically engineered porcine cells, wherein eachgenetically engineered porcine cell of said plurality is characterizedin that at least 97% of the PERV pol genes comprise a geneticallyengineered genomic indel.
 57. The organ of claim 56, wherein 100% of thePERV pol genes comprise a genetically engineered genomic indel.
 58. Theorgan of claim 56, wherein each genetically engineered porcine cellexhibits at least a 1000-fold reduction in PERV transmission asevidenced by co-culturing a human cell with the genetically engineeredporcine cell as compared to co-culturing a human cell with a wild-typeporcine cell.
 59. The organ of claim 58, wherein the human cell isHEK293.
 60. The organ of claim 56, wherein the genetically engineeredgenomic indel is observed by sequencing.
 61. The organ of claim 56,wherein the individual genetically engineered porcine cell containsintact PERV gag and/or env genes.
 62. A method of generating a porcinecell substantially free of porcine endogenous retrovirus (PERV), themethod comprising inserting one or more genetically engineered genomicindels into at least 97% of the PERV pol genes in the porcine cell,thereby inactivating at least 97% of the PERV pol gene.
 63. The methodof claim 62, comprising introducing into the porcine cell at least oneguide RNA (gRNA) comprising a sequence complementary to a portion of aPERV pol gene.
 64. The method of claim 63, wherein the at least one gRNAcomprises SEQ ID NO. gRNA1, SEQ ID NO. gRNA2, or both.
 65. The method ofclaim 63, further comprising introducing into the porcine cell a Casprotein or a nucleic acid encoding a Cas protein.
 66. A progeny cellderived from a genetically engineered PERV-free porcine cell generatedby the method of claim
 62. 67. A composition comprising a plurality ofthe progeny cell of claim 66.